The recently identified ykvM gene family encodes an enzyme belonging to a unique family of nitrile oxido-reductases that catalyze the reduction of 7-cyano-7-deazaguanine to 7-aminomethyl-7-deazaguanine, a transformation never before observed in biology. Nitrile containing compounds are ubiquitous in the pharmaceutical, agricultural, and chemical industries, where they often serve as intermediates in the synthesis of commercially important acids, amides and amines. Biocatalysis has become an important option for catalyzing the conversion of nitriles to acids and amides due to the existence of the enzymes nitrilase and nitrile hydratase, respectively. While the chemical conversion of nitriles to amines suffers some of the same drawbacks as hydrolysis (high energy costs, reactivity of other functional groups, generation of hazardous waste), the absence of a known nitrile oxido-reductase has precluded the application of biocatalysis to this important chemical process. The overall goal of this multi-phase SBIR project is to reengineer the YkvM protein from Bacillis subtilis into an efficient biological catalyst for the industrially important conversion of phenylacetonitrile to phenylethyl amine. Our goal for phase I is to test the feasibility of this concept by creating a mutant enzyme with enhanced activity for the reduction of phenylacetonitrile to phenylethylamine. This will be achieved by the development of a high-throughput assay for screening YkvM mutant libraries, the construction of an appropriate expression system for creating mutant libraries, the application of the parallel and complementary approaches of rational design and directed evolution to create the desired catalyst, and the screening of the libraries to identify the catalyst. The successful realization of the project goals will not only result in the creation of a valuable industrial process, but will also serve as proof-of-concept that this family of enzymes can serve as a platform for the engineering of designed catalysts that carry out the reduction of a variety of commercially important organonitriles. Project Narrative: Relevance: The reduction of nitrile containing compounds to primary amines is a fundamental chemical transformation in the pharmaceutical, commodity, and fine chemical industries. Like many other processes, it is associated with high energy costs, the generation of hazardous waste, and lack of specificity. We propose to use the recently discovered nitrile oxido-reductase enzymes from bacteria as a platform for the design of novel biocatalysts capable of the environmentally benign reduction of targeted nitriles. ? ? ?
